Methods for treatment of HIV and other infections using A T cell or viral activator and anti-retroviral combination therapy

ABSTRACT

Disclosed is a method for treating infection with a pathogen. The method involves administration of: (1) a substance which induces active pathogen replication in a cell latently infected with HIV and (2) an anti-pathogen drug. Also disclosed are methods for expanding CD4+ T cells from peripheral blood mononuclear cells isolated from human subjects in the presence of an antiretroviral drug and for treating HIV infection by infusing the expanded CD4+ cells into HIV-infected patients.

FIELD OF THE INVENTION

[0001] The invention relates to the treatment of Human ImmunodeficiencyVirus (HIV) infection and other infections.

BACKGROUND OF THE INVENTION

[0002] HIV infection leads to progressive deterioration of the immunesystem in most infected subjects. This infection frequently leads to animmune system dysfunction which may culminate in AIDS, a syndromecharacterized by the development of opportunistic infections and cancer(Levy, 1993, Microbiol. Rev., 57:183-289). Cells infected with HIVinclude, e.g., CD4+ T cells, macrophages/monocytes, dendritic cells, andglial cells. The immune system in late stages of AIDS is severelycompromised due to loss or dysfunction of CD4+ T cells (Shearer et al.,1991, AIDS, 5:245-53), macrophages, monocytes, and dendritic cells.(Rosenberg et al., 1989, Adv. Immunol., 46:377).

[0003] Anti-retroviral drugs, such as reverse transcriptase inhibitors,viral protease inhibitors, and viral entry inhibitors have been used totreat HIV infection. (Caliendo et al., 1994, Clin. Infect. Dis.,18:516-24). These treatments can effectively suppress viral productionwhen used in combinations known as HAART (highly active antiretroviraltherapy). However, they are mainly effective in preventing new infectionof uninfected cells. They are generally far less effective ineliminating latent virus from infected cells. Even after two years onHAART, HIV-1 can still be induced, and viral production resume whenHAART is stopped (Finzi et al., 1997, Science, 278:1295-1300; Wong etal., 1997, Science, 278:1291-1295). Hence, HAART likely needs to becontinued indefinitely. This poses significant difficulties. HAARTregimens have many side effects, are difficult to comply with, and areexpensive. Moreover, prolonged treatment with these drugs often leads tothe emergence of drug resistant viral strains (Larder et al., 1989,Science, 246:1155-8; Kellam et al., 1992, Proc. Nat'l. Acad. Sci. USA,89:1934-8; St. Clair et al., 1991, Science, 253:1557-9). Combinationtherapies entailing treatment with two or more drugs which attackdifferent points in the HIV replication cycle delay the emergence ofresistant HIV strains. (D' Aquila, 1994, Clin. Lab. Med., 14:393:422).However, recent data suggest that HIV strains having multi-drugresistance may eventually develop in a significant portion of patientstreated with combination therapy. (Schinazi et al., 1994, Int. AntiviralNews, 2:72-5).

[0004] Many other important infectious pathogens can exist in a latentstate where they are dormant or replicate very slowly. Examples of thesepathogens include retroviruses, e.g., human immunodeficiency virus type2 (HIV-2), human T lymphotropic virus type 2 (HTLV-2); herpesviruses,e.g., Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplextype 1 (HSV-1), herpes simplex type 2 (HSV-2), herpes zoster virus(HZV), herpes virus type 6 (HHV-6), herpes virus type 7 (HHV-7);hepatitis viruses, e.g., hepatitis B (HBV), hepatitis C (HCV), the deltaagent, and hepatitis E, mycobacteria, e.g., M. tuberculosis (MTB), M.avium (MA), M. Leprae; and fungal agents e.g., histoplasmosis,coccidiomycosis, cryptococcus, and pneumocystis. Keeping the infectiouspathogen in latency is desirable when there is no available therapy.However, in many cases, the pathogen is not completely latent andtherapy is required. Unfortunately, while active infection can often becontained by therapy, it is difficult or impossible to attack latentpathogen. Moreover, latent infection can give rise to renewed productionof the infectious microbes when the anti-viral/anti-microbial agents arestopped.

[0005] HIV-2 can cause immunodeficiency similar to HIV-1. HTLV-1 hasbeen shown to cause T cell lymphoma. EBV may cause lymphoma and otherlymphoproliferative diseases. CMV may cause retinitis, hepatitis,pneumonitis, and other systemic illness, especially in immunocompromisedhost. HSV 1 and 2, and Herpes Zoster (HZV) can cause painful vesicles atthe area of infection and occasional meningitis. HHV-6 has beendemonstrated to be present in and may contribute to the pathogenesis incertain subgroups of patients with multiple sclerosis and chronicfatigue syndrome. Nucleoside analogs such as ganiclovir, famciclovir,lamivudine, and ribavirin have been shown to be effective against manyof these infections. These drugs interfere with viral replication, butthey generally cannot attack latent virus. Hence, viral replicationoften resumes when the drugs are withdrawn.

SUMMARY OF THE INVENTION

[0006] The invention features a method for treating HIV infection andother infections. The method entails the administration of: (1) asubstance which induces active pathogen replication in a cell latentlyinfected with the pathogen and (2) an anti-pathogen drug.

[0007] In a preferred embodiment the invention features a method fortreating retroviral, e.g., HIV-1 infection, by administering: (1) asubstance which induces active viral replication in a latently infectedcell and (2) an antiretroviral drug.

[0008] The substance used to induce active viral replication isgenerally a substance which induces the activation or proliferation ofthe latently infected cell, e.g., a T cell, or is substance capable ofinducing viral replication. Mitogenic lectins, such asphytohemagglutinins, can induce the activation or proliferation of humanT cells. Similarly, polyclonal or monoclonal antibodies capable ofbinding T cell surface molecules such as, e.g., α, β or γ, δ T cellreceptors, CD3, CD2, CD4, CD8, CD28, Thy-1, can often induce T cellactivation or proliferation. Additionally, bispecific monoclonalantibodies capable of binding more than one antigen can be used. Forexample, a bispecific monoclonal antibody (BSMAB) having specificity forboth CD3 and CD8, (CD3,8 BSMAB) can induce activation and proliferationof CD4+ T cells. (Wong et al., 1987, J. Immunol., 139:1369-74; Wong etal., 1989, J. Immunol. 143:3403-11, U.S. Pat. No. 5,601,819, allincorporated herein by reference).

[0009] Substances that can activate viral replication directly include:cytokines such as TNF-α and other stimulators of NFκB activity, analogsor fragments of cytokines such as IL-1 and IL-2 and IL-7, andtransactivators encoded by various sequences from various virus, e.g.,herpes virus (HSV, EBV, CMV, HHV-6), HTLV-1, and HBV.

[0010] The anti-retroviral drug used in the methods of the invention canbe any substance which can inhibit, reduce or eliminate retroviralinfection of a cell. Commonly used anti-retroviral drugs include reversetranscriptase inhibitors, protease inhibitors, and inhibitors of viralentry. Reverse transcriptase inhibitors can be nucleoside analogues,e.g., AZT (Zidovudine; Glaxo-Burroughs Wellcome Co., Research TrianglePark, N.C.), ddI (Didanosine; Bristol-Myers Squibb; Wallingford, Conn.),3TC (Glaxo-Burroughs Wellcome), d4T (Stavudine; Bristol-Myers Squibb),or ddC (Zalcitabine; Hoffman-La Roche; Basel, Switzerland); ornon-nucleoside drugs, e.g., Nevirapine (Viramune; Roxane Laboratories;Columbus, Ohio), Delaviridine (Rescriptor; Pharmacia & Upjohn;Kalamazoo, Mich.), Abacavir or Pyridnone (Merck, Sharp & Dohme; Rahway,N.J.). Protease inhibitors which can be used include, e.g., Indinavir(Crixivan; Merck; West Point, Pa.), Ritonavir (Novir; AbbottLaboratories; Abbott Park, Ill.), Saquinavir (Invirase; Roche; PaloAlto, Calif.), Nelfinavir (Agouron Pharmaceuticals; La Jolla, Calif.),and Amprenavir.

[0011] The invention also features a method of treating a patientinfected with HIV, comprising administering to the patient either (a) anamount of a CD4+ T cell mitogen sufficient to induce activation of CD4+T cells and replication of HIV within latently infected CD4+ T cells incombination with a therapeutically effective amount of at least one, butpreferably more than one, anti-retroviral drug, (b) or a direct virusactivator in combination with a therapeutically effective amount of atleast one, but preferably more than one, anti-retroviral drug.

[0012] The invention also features an ex vivo method of expanding CD4+ Tcells from a sample of peripheral blood mononuclear cells (PBMCs)isolated from a human. CD4+ T cells are expanded by culturing PBMCs inan artificial capillary cell culture system in the presence of an amountof a CD4+ T cell mitogen, e.g., CD3,8 BSMAB sufficient to induceactivation of CD4+ T cells and replication of HIV within latentlyinfected CD4+T cells. The invention also features an ex vivo method ofexpanding CD4+ T cells from a sample of peripheral blood mononuclearcells isolated from a human, comprising culturing the peripheral bloodmononuclear cells in an artificial capillary cell culture system in thepresence of a T cell mitogen and at least one anti-retroviral drug.

[0013] The invention also features a method of treating an HIV-infectedpatient by administering to the patient the CD4+ cells grown ex vivo. Bythe methods described above, CD4+ T cells taken from the patient can beexpanded ex vivo to sufficient quantity to transfuse back into thepatient.

[0014] The invention also features a method of treating a patientinfected with HTLV, comprising administering to the patient an amount ofeither (a) a CD4+ T cell mitogen sufficient to induce activation of CD4+T cells and replication of HTLV within latently infected CD4+ T cellsand a therapeutically effective amount of at least one, but preferablymore than one, anti-retroviral drug or (b) a direct virus activator andwith a therapeutically effective amount of at least one, but preferablymore than one, antiretroviral drug. Anti-retroviral drugs can includereverse transcriptase inhibitors, viral protease inhibitors, and viralentry inhibitors (e.g., fragments of viral entry receptors orco-receptors).

[0015] The invention also features a method of treating a patientinfected with members of the herpesvirus family such as EBV, CMX, HSVtype 1, HSV type 2, HHV-6 and HHV-7, comprising administering to thepatient an amount of either (a) a cellular mitogen sufficient to induceactivation of the latently infected cells and replication of the viruswithin the infected cells and therapeutically effective amount of atleast one, but preferably more than one, antiviral drug or (b) a directvirus activator and a therapeutically effective amount of at least one,but preferably more than one, anti-viral drug. The cellular mitogen mayinclude PHA for all cell types, pokeweed mitogen for B cells (EBV), LPS(lipopolysaccharide) for monocytes/macrophages, anti-CD3/TCR for Tcells. Viral activators, some of which may be cellular activators,include IL-6, TNF-α, and GM-CSF. Anti-viral drugs effective against theherpesvirus family include Acyclovir (Glaxo Wellcome), Ganiciclovir(Roche Laboratories), and Famciclovir (SmithKline Beecham).

[0016] The invention also features a method of treating a patientinfected with a hepatitis virus that causes chronic diseases such ashepatitis B, hepatitis C, delta agent, and hepatitis E, comprisingadministering to the patient an amount of either: (a) a cellular mitogensufficient to induce activation of the latently infected cells andreplication of the virus within the infected cells and therapeuticallyeffective amount of at least one, but preferably more than one,anti-viral drug, or (b) a direct virus activator and a therapeuticallyeffective amount of at least one, but preferably more than one,anti-viral drug. The cellular mitogens include PHA for all cell types,LPS (liposaccharide) for monocytes/macrophages, and anti-CD3/TCR for Tcells. Viral activators, some of which may be cellular activators,include IL-6, IFN-Γ, and TNF-α. Anti-viral drugs affective against thehepatitis viruses include: Lamivudine (Glaxo Wellcome), Ganciclovir(Roche Laboratories), Faciclovir (SmithKline Beecham), and IFN-α,(SmithKline Beecham).

[0017] The invention also features a method of treating a patientinfected with a pathogen that has a latent state, e.g., infection with amycobacteria, the method comprising administering to the patient either(a) an amount of cellular mitogen sufficient to induce activation of thelatently infected cells and replication of the infectious pathogen and atherapeutically effective amount of at least one, but preferably morethan one, anti-infection drug or (b) a direct activator of theinfectious pathogen and a therapeutically effective amount of at leastone, but preferably more than one, anti-infection drug. Suitablecellular mitogens include: PHA for all cell types, LPS (liposaccharide)for monocytes/macrophages, and anti-CD3/TCR for T cells. Activatorsinclude corticosteroid, TNF-α, and IL-2. In some cases, activators ofthe microorganism may also act as cellular activators. Drugs which maybe used to treat mycobacterial infection include isoniazid, rifampin,clarithromycin, and ethambutol.

[0018] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described below. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. In the case of conflict,the present document, including definitions, will control. Unlessotherwise indicated, materials, methods, and examples described hereinare illustrative only and not intended to be limiting.

[0019] Various features of the invention will be apparent from thefollowing detailed description and from the claims.

BRIEF DESCRIPTION OF THE DREAWINGS

[0020]FIG. 1 is a graph depicting proliferation of CD4+ T cells inducedby culturing PBMCs isolated from non—HIV infected subjects in thepresence of CD3,8 BSMAB and IL-2.

[0021]FIG. 2 is a graph depicting proliferation of CD4+ T cells inducedby culturing PBMCs isolated from HIV-infected subjects in the presenceof CD3,8 BSMAB and IL-2.

[0022]FIG. 3 is a graph depicting the rapid increase in HIV productionwhen CD4+ T cells are cultured with CD3, 8 BSMAB and IL-2 in the absenceof retroviral drugs.

[0023]FIG. 4 is a table of HIV-1 infected subject characteristics andmaximum CD4+ T cell expansion.

[0024]FIG. 5 is a table of HIV-1 p24 antigen production.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The invention is directed to a method of treating infection witha pathogen by administering a compound which provides a strongactivation stimulus that activates all or nearly all the pathogen fromthe latent state and concurrently administering one or moreanti-pathogen drugs that can inhibit pathogen replication or viability.Under these conditions, infected cells are killed by the replicatingpathogen but infection of uninfected cells is largely prevented by theanti-pathogen drug. Thus, by inducing the death of latently infectedcells, the method of the invention can substantially reduce thereservoir of latently-infected cells present in the patient.

[0026] The invention is directed to a method of treating HIV infectionby administering a compound which provides a strong activation stimulusthat activates all or nearly all the virus from the latent state andconcurrently administering one or more anti-retroviral drugs that caninhibit viral replication. Under these conditions, infected cells arekilled by the replicating virus but infection of uninfected cells islargely prevented by the anti-retroviral drugs. Any viruses remainingextracellularly are rapidly degraded because HIV is unstable in thefluid phase. Thus, by inducing the death of latently infected cells, themethod of the invention can substantially reduce the reservoir oflatently-infected cells present in the patient.

[0027] HIV replication in latently infected CD4+ T cells can be inducedby activating the cells. Similarly, HIV in latently infected macrophagesor monocytes can be activated by the cytokines such as tumor necrosisfactor (TNF-α)that are released by activated T cells. The concurrentpresence of the cell stimulus and anti-retroviral therapy is importantbecause activation in the absence of anti-retroviral therapy potentiallycan lead to acceleration of the infection.

[0028] Concurrent In Vivo Administration of a T cell Mitogen and One orMore Anti-retroviral Drugs to an HIV-infected Subject

[0029] HIV-infected subjects are treated with a therapeuticallyeffective amount of a T cell mitogen and at least one anti-retroviraldrug. Those skilled in the art can select an appropriate therapeuticregime employing one or more anti-retroviral drugs. For example,combinations and dosages of anti-retroviral drugs can be determined frompublished recommendations (Carpenter et al., 1997, Anti-retroviralTherapy for HIV Infection in 1997-Updated Recommendations of theInternational AIDS Society-USA Panel, J. American Medical Assoc.,277:1962). Suitable T cell mitogens include any substance which has theability to activate T cells or cause their proliferation. For example,mitogenic lectins (e.g., PHA), mitogenic antibodies, (e.g., anti-CD3specific antibodies), mitogenic bispecific antibodies (e.g., CD3,8BSMAB), lymphokines (e.g., IL-2) and cytokines (e.g., TNF-α).Appropriate dosages of T cell mitogens to be administered to anHIV-infected person can be determined by one skilled in the art. Forexample, concentrations of T cell mitogens known to cause theproliferation of T cells in vitro, can be extrapolated to determine thedosage required to generate in vivo T cell activation or proliferation.

[0030] For example, 0.02-0.2 μg/ml of CD3,8 BSMAB causes T cellproliferation in an in vitro assay. Accordingly, CD3,8 BSMAB can beadministered at, e.g., 4 to 40 mg/kg/day to a human subject forapproximately, e.g., 3-7 days until the subject's plasma concentrationof CD3,8 BSMAB is between 0.02-0.2 μg/ml. Similarly, the dose of T cellmitogen necessary to achieve T cell activation or proliferation in ahuman subject can be extrapolated from data obtained from animalexperiments. The dose of T cell mitogen should be sufficient to induceactivation of CD4+ T cells and replication of HIV within latentlyinfected T cells.

[0031] In one embodiment, HIV-infected subjects are treated with: (1) acombination of AZT (zidovudine) 200 mg 3×/day, 3TC (lamivudine) 150 mg2×/day, and Indinavir 800 mg/day for a minimum of one week; (2) CD3,8BSMAB at 4-40 μg/kg/day for 3 to 7 days (until plasma level is 0.02-0.2μg/ml); and (3) IL-2 at 106 units/m² daily for one month. The dosage ofIL-2 is then decreased gradually to zero over two months to threemonths. Of course, the T cell mitogen and the anti-retroviral drugs neednot be co-administered. However, a therapeutic amount of theanti-retroviral drugs should be present during stimulation or T cellproliferation.

[0032] Throughout the treatment, the subjects can be monitored foradverse effects of the treatment protocol, e.g., vital signs, liverenzymes, renal function, and glucose levels. T cell activation can beassessed by CBC with differential, IL-2 receptor or HLA-DR expression.Patient blood samples can be tested for HIV by several methods known tothose skilled in the art. For example, levels of anti-HIV antibody andviral proteins can be determined by standard commercial kits (e.g.,ELISA or RIA). Polymerase chain reaction (PCR) can be used to assess thepresence of HIV nucleic acid in biological samples. PBMCs can beharvested from the treated subjects at two week intervals and tested forinfectious HIV by co-culturing with known non-infected CD4+ cells.Infection of previously non-infected cells would indicate thatinfectious HIV was present in the subject. The foregoing assays can alsobe used to test lymph node samples from the subject for infectious HIV.

[0033] Ex Vivo Expansion of CD4+ T Cells from PBMCs Isolated from HumanSublects

[0034] CD4+ T cells can be expanded from a sample of PBMCs isolated froman HIV-infected or non-HIV infected subjects according to the method ofWilson et al. (1995, J. Infect. Dis., 172:88-96). PBMCs can be isolatedfrom a human by methods well known in the art. For example, a sample ofblood can be removed from a human via a syringe and needle and placedinto a container containing anti-coagulants. PBMCs can be isolated bycentrifuging the sample through a ficoll diatrizoate gradient (Sigma,St. Louis, Mo.).

[0035] CD4+ T cells can also be expanded from a sample of PBMCs isolatedfrom an HIV-infected or non-HIV infected subject using an artificialcapillary system (Knazek et al, 1972, Science, 178:65-67; Bresler etal., 1993, Repetitive Expansion of Large Numbers of CD8+ Lymphocyteswithin an Artificial Capillary System, Abstract/AAI & CIS ConjoinedAnnual Meeting). Artificial capillary systems are availablecommercially, e.g., CELLMAX® artificial capillary system (Cellco, Inc.,Germantown, Md.).

[0036] In one embodiment of the invention, the CELLMAX® artificialcapillary culture system (Cellco) is primed by pre-infusion for 24-72hours with 100 ml of Cellgro Complete Serum Free Medium (Mediatech)supplemented with 1% human AB serum (Sigma), gentamicin (Gibco) 50μg/ml, and cefoxitin (Merck) 50 μg/ml (designated complete medium). Theantibiotics are used to inhibit bacterial growth should the rarecontamination occur. On day zero, 3-5×10⁷ PBMC (only a fraction of whichwill be CD4+ T cells) are suspended in 12 ml of complete mediumsupplemented with rIL-2 (Hoffman-LaRoche) 100 U/ml and CD3,8 BSMAB 6μg/ml. The triple anti-retroviral combination of either (a) AZT(Zidovudine, Glaxo-Burroughs Wellcome Co.) 1 μM + ddI (Didanosine,Bristol-Myers Squibb) 10 μM + nevirapine 0.1 μM or (b) AZT + 3TC(Glaxo-Burroughs Wellcome Co.) + Indinavir (Merck) 0.4 μM are added.Other combinations can be selected based on analysis of the drugresistance pattern determined in screening plate cultures.

[0037] The cell suspension is then inoculated directly into theextracapillary space (ECS) of the Cellmax artificial capillary culturecartridge. Next, 150 ml of the complete medium, supplemented with theIL-2 and the triple anti-retroviral agents, is added to the feedingreservoir. The whole system is then placed into a designated incubatorset at 37° C. and 5% CO₂. Perfusion rate is set at 50 ml/min for thefirst 7-10 days of culture. On day 3, the CD3,8 BSMAB is largely removedfrom the ECS of the cartridge by replacing 10 ml of the ECS medium withthe IL-2/triple anti-retroviral drug supplemented complete medium.

[0038] The rate and cell growth and consumption of nutrient is followedby measuring the glucose consumption with the Accu-Chek Advantageglucometer (Boehringer Mannheim) after diluting the medium 1:1 with PBS.When the glucose concentration drops to approximately 100-150 mg/dl, thefeeding medium in the reservoir bottle is increased to 300 ml. With thenext equivalent drop in glucose, the reservoir medium is increased to500 ml and the perfusion rate increased to 100 ml/min. At day 21 (or day19), the feeding medium is changed to complete medium supplemented withIL-2 but without anti-retroviral drugs. When the glucose concentrationagain drops to 150 mg/dl (at the 500 ml stage), the cells are harvestedand counted. During the harvest, ¾ to {fraction (7/8)} of the cells areremoved by 3-4 successive vigorous washes, leaving {fraction (1/4)}, to⅛ of the cells in the cartridge. Approximately 0.8-1.8×10⁹ cells areremoved by each harvest. The remaining cells are fed with newIL-2-supplemented complete medium and be allowed to grow.

[0039] Repeated harvesting can be performed with every other exchange ofthe 500 ml feeding medium until the cultured cells no longer grow(defined as decreasing number of viable cells for more than two weeks)or when the total numbers of CD4+ T cells generated has reached 10²¹cells (the equivalent of approximately 47 divisions).

[0040] Expansion for each harvest is calculated by the total number ofCD4+ T cells in the cartridge at the time of harvest (number of cellsremoved + the calculated number of cells remaining in the cartridge[estimated by the 12 ml volume of the ECS x cell concentration of thelast wash volume])/starting number of CD4+ T cells. The total cellexpansion is calculated by the multiplying the fold expansion of eachharvest. The total number of expanded cells is then calculated bymultiplying the total number of cells after expansion by the number ofCD4+ T cells at the initiation of culture. Cells removed during harvestcan be phenotypically typed and/or frozen in 10×10⁶ cell aliquots in,for example, liquid nitrogen, after each harvest.

[0041] The frozen cells can be thawed in batches for immunobiologicalassays. The ECS supernatant removed during the cell harvest can also befrozen and thawed for batched p24 assays. Collected supernatants arediluted by the addition of {fraction (1/10)} volume of 5% Triton X−100(DuPont), and stored at −20° C. until run using a standard DuPontenzyme-linked immunoabsorbent assay (ELISA) kit for the detection ofHIV-1 p24 antigen. Samples from individual subjects can be stored andrun in batches. A standard curve can be generated in each assay, andthose values which exceeded the reactive threshold of 0.05 plus the meanoptical density of the negative controls (equivalent to 4 standarddeviations above the mean) are considered to be positive.

[0042] Treatment of HIV-infected Patients with Ex Vivo Expanded CD4+ Tcells

[0043] For infusion of ex vivo expanded CD4+ cells, PBMCs can beisolated from a human subject and expanded as described above. Theexpanded CD4+ cells can be removed from cell culture, washed free ofmedium, placed in a pharmaceutically acceptable carrier (e.g. sterilesaline, human serum, or human blood), and infused into the subjectintravenously by methods known in the art. Preferably, the PBMCs to becultured for expansion in to CD4+ T cells are autologous to the subjectto be infused.

[0044] Administration of CD3,8 ESMAB to PBMCs Isolated from HIV-infectedPatients

[0045] In the absence of anti-retroviral drugs, the addition of a highconcentration of CD3,8 BSMAB in the presence of IL-2 to a culture ofPBMCs isolated from HIV-infected subjects led to the depletion of CD8+ Tcells and an expansion of CD4+ T cells. The percent of CD8+ cells in theculture increased with lower concentrations of CD3,8 BSMAB. Atconcentrations between 0.2-2.0 μg/ml, there was a net increase in bothCD4+ T cells and CD8+ T cells, with greater expansion of the CD4+ Tcells. In comparison, in response to CD3,8 BSMAB alone, the CD4+ T cellswere activated to proliferate and express CD25 (interleukin-2 receptor).The stimulation of the PBMCs in this manner turn leads to a massiveincrease in HIV replication, as measured by the marked elevation of p24HIV protein levels in the harvested tissue culture supernatant (FIG. 5).Absent antiretroviral drugs, this massive activation of HIV willeventually cause a sharp decrease in the CD4+ T cell population.

[0046] Adding phytohemagglutinin (PHA) or the anti-CD3 monoclonalantibody, 12F6, to the culture of PBMCs similarly induced increased HIVreplication. In this case, however, both CD4+ and CD8+ T cells wereactivated. No selective outgrowth of the CD4+ T cells occurred, andlater in the culture the CD4+ T cell population rapidly declined.

[0047] The foregoing experiments were also performed in the presence ofan effective amount of the anti-retroviral drug combination: 1 μMZidovudine (AZT), lOAM Didanosine (ddI), and 0.6 μM pyridnone. Additionof CD3,8 BSMAB (1.5-3.0 μg/ml) to the culture of PBMCs isolated fromHIV-infected subjects induced preferential outgrowth of the CD4+ Tcells. Initially, a mild increase in virus replication occurred asindicated by a slight elevation of p24 in the culture supernatant. Thiswas followed by rapid elimination of HIV in 9 out of 12 cultures asevidenced by absence of detectable HIV replication (measured by p24antigen detection) and the inability of these cells to infect othercells in co-culture experiments even after the antiretroviral drugs wereremoved (FIG. 5). Using the very sensitive polymerase chain reaction(PCR) technique, a very low level of HIV nucleic acid was detected inthese cultures. Since no viral proteins were detected in these culturesand the treated cells did not infect co-cultured cells even withrepeated stimulation, this PCR-detected HIV nucleic acid likelyrepresents integrated incompetent viral genomic sequences that arosefrom mutated strains or viral genomic sequences integrated at locationswhere they cannot be activated to replicate.

[0048] A single course of CD3,8 BSMAB, in the presence ofanti-retroviral drugs, induced proliferation of CD4+ T cells from PBMCsof HIV-infected subjects

[0049] PBMCs (5×10⁷) isolated from HIV-infected subjects were placed ina hollow fiber cartridge cell culture system (CELLMAX®, Cellco, Inc.), asingle course of CD3,8 BSMAB was added for 4 days. The cultures wereperiodically assessed over a period of just over 200 days. Throughoutthis period there was a continuous expansion of CD4+ T cells. Takinginto account fractionation of cultures to prevent overgrowth, afterbetween 50 to 200 days, approximately 10¹⁰ cells to >10¹⁷ cells could beproduced from the initial inoculum of PBMCs.

[0050] We proceeded to examine whether the PBMC of HIV-1 infectedsubjects can also give rise to a continuously expanding CD4+ T cells ina CD3,8/IL-2 artificial capillary culture system. Triple anti-retroviraltherapy (AZT, ddI, and nevirapine) was used for the first 19 days inculture and then replaced by fresh medium with IL-2 but without theanti-retroviral drugs. The PBMC from six HIV-1 subjects were studied todate (FIG. 4). Their growth pattern is plotted in FIG. 2.

[0051] Subject 161j is a long term nonprogressor with seroconversion 16years ago. He is asymptomatic and has 1400 CD4+ T cell counts (Table I).Starting with 107 cells, his CD4+ T cells expanded to 3×10¹⁸ cellsbefore plateauing. This represented an average expansion of 3×10¹¹-fold,dividing an average of 38 times for each cell, comparable to those ofnon-infected subjects. There was no detectable p24 production. Subject1436 g had a circulating CD4+ T cell count of 350/μl and wasasymptomatic at the time of testing. In the presence of CD3,8 BS,AB forfour days and triple anti-retroviral therapy for 19 days, his PBMCculture similarly rapidly became predominantly CD4+ (95%) and expandedexponentially, reaching 3×10¹⁴ cells by the 80th day in culture. Thisrepresented an expansion of 3×10⁷ fold (an average of 25 divisions) whenthe culture was arbitrarily stopped while still expanding exponentially.

[0052] Transfer of part of the harvested cells at the 10¹² stage ofexpansion to a new cartridge resulted in a quick re-establishment ofgrowth in a pattern that paralleled the parent cartridge after a 10 daylag period. This demonstrated that the theoretical total expansion isachievable if all the harvested cells are placed into new cartridges.The p24 level was low by the first week and declined progressively overthe next 30 days. The small amount of p24 detected likely representeddilution of the initial production as the level declined to belowdetectable levels in the absence of further anti-retroviral therapy.Parallel CD3,8 BSMAB/IL-2 treated cultures in the absence ofanti-retroviral therapy produced rapid p24 production.

[0053] Subject 011441 g's peripheral CD4+ T cell count was 400/μl at thestart of the culture. His PBMC culture also rapidly became predominantlyCD4+ T cells. There was a slight delay in the early phase of growth, butthe culture eventually reached exponential growth. The culture wasstopped at 10¹² cells arbitrarily by the 75th day of culture while thecells were still in exponential growth (10⁵ fold expansion and anaverage of 16.7 divisions per cell). Harvested cells that weretransferred picked up a growth rate that paralleled the originalculture. The p24 production was low by the first week and continued todecline over the next 30 days in similar pattern to the other. SubjectSB had a peripheral CD4+ T cell count of 300 at the time of the culture.His PBMC gave rise to 10¹² cells (10⁵ fold expansion and an average of16.8 divisions per cell) by the 60th day and then plateaued between the60-90th day. Subject 012215e's peripheral CD4+ T cell count was also300/μl. His culture took off rapidly but then plateaued by the 30th dayat only 2.2×10¹⁰ cells (4×10³ fold expansion and an average of 12divisions per cell). This early plateau and limited expansion was notdue to viral production as the p24 production was low and declinedgradually in similar fashion to the CD4+ T cell cultures of the othersubjects (FIG. 3). The transferred cells likewise plateaued at an earlypoint and paralleled the rate of growth of the parent culture.

[0054] The results presented in FIG. 3 demonstrate the rapid increase inHIV production when the CD4+ T cells were stimulated by CD3,8 + IL-2 inthe absence of anti-retroviral drugs. The results presented in thisfigure also demonstrate that the virus reproduced at a constant lowlevel for several weeks followed by a decline to undetectable level whenthe CD4+ T cells were stimulated by CD3,8 + IL-2 in the presence ofeffective anti-retroviral drug combination. No virus replication wasdetectable even after the anti-retroviral drugs were removed, utilizingan ultrasensitive RNA PCR that can detect down to 40 copies per cc.

[0055] Subject 011430j had a CD4+ T cell count of 170/μl at the start ofthe culture. His CD4+ T cells showed the same initial rapid expansion to10³ cells (10³ fold expansion and an average of 10 divisions per cell)by the 40th day but then rapidly declined.

[0056] In this experiment, no restimulation was used after the initialround of expansion. However, the expanded, virus free CD4+ T cells canbe further expanded with additional round of stimulation in the presenceof fresh accessory cells.

[0057] Importantly, the expanded CD4+ T cells were free of competentHIV. Since the total number of CD4+ T cells in the human body isapproximately 10¹¹-10 ¹², these experiments demonstrated that one canobtain more than enough number of CD4+ T lymphocytes to completelyreplace CD4+ T lymphocytes in the body with reserve for furtherexpansion, starting with a relatively small amount of peripheral bloodmononuclear cells (including CD4+ T lymphocytes).

[0058] In one experiment performed using the above protocol, PBMCs wereisolated from three non-HIV infected subjects (Subjects A, B, and C) andthen incubated with CD3,8 BSMAB and IL-2 in the artificial capillaryculture cartridge. After 4 days, medium was exchanged with fresh mediumcontaining IL-2 and CD3,8 BSMAB. With periodic replacement of themedium, the cells divided rapidly. When the culture contained between1-2×10⁹ cells, ¾ to ⅞ of the cells were harvested. The cells remainingin the cartridge continued to grow rapidly and each culture could beharvested repeatedly. The harvested cells were analyzed and frozenperiodically.

[0059] The proliferation of T cells in cultures of PBMC from threeSubjects A, B, and C are illustrated in FIG. 1. The CD4+ T cells fromsubject A expanded steadily in response to a single stimulation by CD3,8BSMAB, giving rise to 10¹⁹ cells by the 78th day of culture. The culturewas stopped arbitrarily at that point, while the cells were still inexponential phase of growth. Similarly, the CD4+ T cells from subject Balso expanded exponentially to about 10¹⁵ cells, when the culture wasarbitrarily stopped. In comparison, the CD4+ T cells from subject Cexpanded to 10^(l3) cells, plateaued, and then gradually declined.

[0060] Microscopic examination of the cells harvested throughout thedifferent periods of culture all showed the elongated shapes typical ofT cell blasts. By flow cytometry, all the cultures rapidly become 94-98%CD4+ T cells without detectable CD8+ T cells. In the early to mid phasesof expansion, approximately half the cells express predominantly CD45RAand the other half express predominantly CD45RO. In the late phase ofexpansion, there was a gradual shift toward CD45RO expression in somecultures. This shift occurred at a much later stage of expansion than isexpected from the literature, where the shift toward the “memory”phenotype (CD45RO) is reported to occur soon after mitogen-inducedexpansion. The CD4+ T cells expressed a high level of the IL-2Rα chain(CD25) throughout the culture, consistent with maintenance of activatedstatus with responsiveness to IL-2. When pulsed with ³H-thymidine, theCD4+ T cells harvested throughout the different phases of expansionshowed high level of DNA synthesis. These cells were not permanentlytransformed as they died when placed in standard culture flasks forseveral weeks in the presence of IL-2 (death occurred more quickly ifIL-2 was withdrawn). The lack of transformation is further supported bythe eventual decline in division of the cells of subject C even whenmaintained optimally in the artificial capillary culture cartridge. Byflow cytometry, 0.2-2.0% of the harvested cells throughout the differentphases were CD4+CD3−, a phenotype suggestive ofmacrophage/monocytes/dendritic cells. This observation is consistentwith the rare adherent cells with pseudopods that were seen in someharvests.

[0061] Activation of HIV Renders the Virus Susceptible toAnti-Retroviral Therapy.

[0062] Since AZT, ddI, pyridinone, and the like inhibit HIV replicationby blocking reverse transcription of HIV RNA, they are not effective ineliminating latent integrated virus from infected cells. Hence,elimination of competent HIV from the cultures indicated that thelatently infected cells were in fact killed, most likely by the activelyreplicating virus. The small transient rise in the p24 antigen, observedearly in the culture, likely reflected this initial replication. Thepresence of the anti-retroviral combination therapy prevented the newlyreleased virus from taking hold in the expanded CD4+T cells. Hence, thesuccess of these drug combinations is dependent on the activation of thelatent virus to eliminate the virus.

[0063] Activation of T cells Eliminates HIV from CD4+ T Lymphocytes andMonocytes.

[0064] In these experiments, peripheral blood mononuclear cells (PBMC)from HIV-infected subjects were used. PBMC not only have variouslymphocytes subsets (CD4+, CD8+, etc.), they also contain substantialnumber of monocytes and dendritic cells. Elimination of HIV from theabove cultures demonstrated that HIV was eliminated from monocytes anddendritic cells as well as in T cells. This elimination of HIV inmonocytes and dendritic cells was not due to dying off of monocytes asthe hollow fiber cartridge cultures continue to contain small amount ofmonocytes even in late culture. Since CD3,8 BSMAB bind to and activate Tcells rather monocytes and dendritic cells, perhaps the activated Tcells secrete cytokines such as TNF-α and IFN-Γ which in turn activatethe HIV replication cycle in latently-infected monocytes and dendriticcells.

[0065] Similar procedures can be modified to treat patients infectedwith HTLV, herpesvirus family (EBV, CMV, HSV, HHV, HZV), hepatitis(hepatitis B, hepatitis C, delta agent, hepatitis E), mycobacteria (M.tuberculosis and M. leprae), and other infections with latent phase.

What is claimed is:
 1. A method of treating a patient infected with HIV,comprising administering to said patient an amount of a T cell mitogensufficient to induce activation of CD4+ T cells and replication of HIVwithin latently infected T cells and a therapeutically effective amountof an anti-retroviral drug.
 2. The method of claim 1, wherein saidpatient is treated with a therapeutically effective amount of at leasttwo anti-retroviral drugs.
 3. The method of claim 1 or 2, wherein the Tcell mitogen is a lectin or an antibody.
 4. The method of claim 1 or 2,wherein the T cell mitogen is a bispecific monoclonal antibody havingspecificity for CD3 and CD8.
 5. A ex vivo method of expanding CD4+ Tcells from a sample of peripheral blood mononuclear cells isolated froma human, comprising culturing said peripheral blood mononuclear cells inan artificial capillary cell culture system in the presence of a T cellmitogen and an anti-retroviral drug.
 6. The method of claim 5, whereinsaid culturing takes place in the presence of at least twoanti-retroviral drugs.
 7. The method of claim 5 or 6, wherein the T cellmitogen is a lectin or an antibody.
 8. The method of claim 5 or 6,wherein the T cell mitogen is a bispecific monoclonal antibody havingspecificity for CD3 and CD8.
 9. A method of treating a patient infectedwith HIV, comprising administering to said patient CD4+ cells expandedaccording to the method of claim
 5. 10. A method of treating a patientinfected with HIV, comprising administering to said patient CD4+ cellsexpanded according to the method of claim
 6. 11. A method of treating apatient infected with HIV, comprising administering to said patientCD4+cells expanded according to the method of claim
 7. 12. A method oftreating a patient infected with HIV, comprising administering to saidpatient CD4+cells expanded according to the method of claim
 8. 13. Amethod of treating a patient infected with HIV, comprising administeringto said patient an activator of viral replication and a therapeuticallyeffective amount of an anti-retroviral drug.
 14. A method of treating apatient infected with a retrovirus, comprising administering to saidpatient an amount of cellular mitogen sufficient to induce bothactivation of cells latently infected with said retrovirus andreplication of the retrovirus and a therapeutically effective amount ofan anti-retroviral drug.
 15. A method of treating a patient infectedwith a retrovirus comprising administering to said patient an activatorof retroviral replication and a therapeutically effective amount of ananti-retroviral drug.
 16. A method of treating a patient infected with avirus, comprising administering to said patient an amount of a cellularmitogen sufficient to induce both activation of cells latently infectedwith said virus and replication of said virus and a therapeuticallyeffective amount of an anti-viral drug.
 17. A method of treating apatient infected with a virus comprising administering to said patientan amount of a viral activator sufficient to induce viral replicationand a therapeutically effective amount of an antiviral drug.
 18. Themethod of claim 16 or 17 wherein said virus is a member of the hepatitisvirus family.
 19. The method of claim 16 or 17 wherein said virus is amember of the herpes virus family.
 20. A method of treating a patientinfected with a pathogen, comprising administering to said patient anamount of a cellular mitogen sufficient to induce both activation ofcells latently infected with said pathogen and replication of saidpathogen and a therapeutically effective amount of an anti-pathogendrug.
 21. A method of treating a patient infected with a pathogencomprising administering to said patient an amount of an activatorsufficient to induce pathogen replication and a therapeuticallyeffective amount or an anti-pathogen drug.